Approaches to Discriminate Naturally Induced Foxp3+ Treg cells of Intra- and Extrathymic Origin: Helios, Neuropilin-1, and Foxp3RFP/GFP

CD4+ regulatory T (Treg) cells expressing the transcription factor fork head box P3 (Foxp3) prevent catastrophic autoimmunity and maintain immune homeostasis throughout life, and are being increasingly implicated in non-immune functions, such as the control of metabolic and regenerative processes in mice and humans. Early studies have attributed a major role of the thymus and intrathymic Foxp3+ Treg lineage commitment (referred to as ‘tTreg’ cells) in the establishment and maintenance of the mature Foxp3+ Treg cell pool residing in peripheral lymphoid tissue. In addition, numerous experimental modalities have been shown to instruct Foxp3+ Treg cell commitment in peripheral, initially naive CD4+Foxp3- T cells, which includes the induction of a Foxp3+ Treg cell phenotype and suppressor function by TGF-β in vitro (‘iTreg’ cells) and by sub-immunogenic T cell receptor stimulation in vivo (‘pTreg’ cells). This led to the hypothesis that, under physiological conditions, the induction of pTreg cells may also contribute to the peripheral Foxp3+ Treg cell compartment in the steady state of nonmanipulated, immunocompetent mice. However, until recently, studies on developmental Foxp3+ Treg cell heterogeneity have been hampered by the lack of suitable markers to discriminate naturally induced tTreg and pTreg cells. Here, we provide an overview of recently proposed approaches to track such developmental sub-lineages, with a particular emphasis on Helios, Neuropilin-1, and Foxp3RFP/GFP mice, in which Foxp3RFP+ tTreg and pTreg cells are stably marked by differential GFP expression.